The overall goal of our research program is to unravel the pathogenic mechanisms in human hereditary retinal degenerations, contribute to the design of mechanism-appropriate therapies, and develop means to test their safety and efficacy. In the current proposal, we focus on recessive retinal degenerations (RD) caused by mutations in the ABCA4 gene, diseases which have attracted recent interest as potential candidates for gene replacement therapy. Patients with ABCA4-RD commonly have diagnoses of Stargardt disease or cone-rod dystrophy but show very wide phenotypic heterogeneity. Our recent findings elucidated the source of this heterogeneity by estimating disease severity caused by each individual ABCA4 allele. Unexpectedly, a substantial fraction of non-truncating alleles were shown to cause greater disease severity than truncating alleles. This finding cannot be explained by a pure loss-of-function pathogenic mechanism and suggests the existence of an additional gain-of-function mechanism in this recessive disease. Aims 1 and 2 will test this hypothesis in order to define genotypes not amenable to gene replacement therapy. In Aim 1, we use an independent cohort of patients to validate the severity estimates of disease-causing ABCA4 alleles. In Aim 2, we test the ability of gene therapy to arrest photoreceptor degeneration in models of complete loss of function and combined loss- and gain-of-function. A recently developed abca4-/-rdh8-/- mouse with severe photoreceptor degeneration will be used as the first model. As for the second model, we will use the human allele severity data to generate a knock-in mouse with complex pathogenesis. Lastly, in Aim 3 we evaluate hypotheses about the disease stage to include in future gene therapy trials such that there is an optimal combination of remaining photoreceptors to treat and a rapid progression of disease to detect. The studies proposed in this application are not only prerequisites for the safe conduct of ABCA4 gene therapy trials but also define a path to follow for other recessive retinal degenerations. PUBLIC HEALTH RELEVANCE: Incurable vision loss is the result of most inherited retinal degenerations with one exception: patients with a rare molecular form caused by RPE65 mutations were recently treated with gene therapy and they showed substantial restoration of vision. It is likely that this promising treatment approach will soon be applied to more common conditions. Mutations in ABCA4 gene cause one of the most common forms of retinal degeneration and the goal of the proposed research is to define the subset of patients with ABCA4 disease who are likely to benefit from gene replacement therapy.